Fig. 4. PKC δ controls IGF-1R overexpression in PCA.

(A) Total cell lysates from AsPC-1 cells untreated (control) or treated with 7 or 10 nM of the PKC inhibitor Goe6983 were resolved by SDS-PAGE and analyzed by immunoblotting with anti-IGF-1R 2C8 (IGF-1R) and anti-phospho mTOR (phos-mTOR). β-actin served as control protein. Up to a concentration of 7nM Goe6983 blocks specifically calcium-dependent PKC α and β. Up to 10nM Goe6983 also inhibits novel PKC’s, including PKC δ and in higher concentrations than 10nM to atypical PKC’s including PKC ζ. β-actin served as control protein. Same extracts were immunoblotted with anti-Phos-S6K, -S6K, -Phos-4E-BP and -4E-BP antibodies. (B) Total cell lysates from AsPC-1 cells transiently transfected with 0.5, 1.0 and 1.5 µg of dominant negative PKC δ (dn-PKC-δ) or empty vector (control) were analyzed by immunoblotting with anti-IGF-1R 2C8 (IGF-IR) and anti-phospho-mTOR (phos-mTOR). β-actin served as control protein. Same extracts were immunoblotted with anti-Phos-4E-BP and -4E-BP antibodies. (lower-panel) Total cell lysates from AsPC-1 cells transiently transfected with 0.5 and 1.0 µg of dominant negative PKC-ζ or empty vector (control) were resolved by SDS-PAGE and analyzed by immunoblotting with anti-IGF-1R 2C8 (IGF-IR) and anti-phospho-mTOR (phos-mTOR). β-actin served as loading control. (C) IGF-1R mRNA extracted from AsPC-1 cells transiently transfected with 1.0 and 1.5 µg of dominant negative PKC-δ (dn-PKC-δ) or empty vector (control) was analyzed by RT-PCR using beta-actin as control. (D) Total cell lysates from AsPC-1 cells untreated (control), 50 µM TATFLAG peptide (control) and treated with 20 and 50 µM of TATFLAGVHL-peptide, were resolved by SDS-PAGE and analyzed by immunoblotting with anti-phospho-mTOR (phos-mTOR) and anti-mTOR (mTOR). TATFLAGVHL peptide (107–122) binds directly to PKC-δ and blocks its kinase activity.